273

A MINI-REVIEW ON BIOFUELS FROM CHLAMYDOMONAS REINHARDTII

Asmaa Missoum*

Address(es): Department of Biological and Environmental Sciences, College of Arts and Sciences, Qatar University, Doha City, Qatar.

*Corresponding author: amissoum93@gmail.com ABSTRACT

Keywords: Chlamydomonas reinhardtii, Biofuel, Microalgae, TAGs, TAPP, EER

INTRODUCTION

A “biofuel” is a type of fuel that is derived from living organisms such as plants,

fungi, or algae. Research on these types of fuels has evolved since the past three decades as they offer an alternative source of sustainable energy compared to

fossil fuels, which are depleting and causing huge environmental concerns such

as the green house gases (Pandey et al., 2014). Because extracting sources of energy such as biofuel from living organisms requires series of processes,

biotechnology play an essential role in this field (Radakovits et al., 2010). This

technology involves the utilization and manipulation of biological processes in

microorganisms for industrial purposes such as the production of hormones,

antibiotics, and biofuels. Few of the sources of biodiesel that caught many

researchers’ attention due to its various advantages are algal species (Lam and

Lee, 2012).

Algae are photosynthetic microorganisms that either belongs to prokaryotes or

eukaryotes. They range from unicellular structure including some filamentous and colonial algae (Microalgae) to simple multi cellular structure (Macroalgae).

There are more than 50,000 of microalgae species living in various ecosystems

including terrestrial and aquatic ones (Brennan, 2010). The cultivation of microalgae as source of biomass has many advantages such as they have higher

capacity to capture sunlight and use it for photosynthesis in order to produce organic compounds (Mata et al., 2010). Moreover, microalgae produce higher

amounts of polymers, pigments, carbohydrates, lipids, and proteins when they

are subjected to chemical and physical stress conditions. Compared to other microorganisms, many microalgae possess simple cellular division cycle without

a sexual stage, which allows them to grow rapidly and eventually produce high

biomass yield. Finally, because microalgae live in all kinds of environments differing in temperature, salinity, and light intensities, it is easier to harvest ones

with prefered properties such as tolerating extreme conditions (Pandey et al.,

2014). Thus, they do not need mass areas of lands or fresh waters as they can even grow in sewage or waste water. Other facts that must be taken into

consideration are: microalgae-based fuels do not compete with food supply such

as corn and soybeans based biofuels, are renewable, and can be carbon reducing.

In fact, generating 100 tons of algae biomass is equivalent to removing about 183 tons of carbon dioxide from the atmosphere (Pandey et al., 2014).

Chlamydomonas reinhardtii is considered one of the best candidates for biofuel

production. Some of the various reasons are that this model organism has been the lead in development of molecular tools for engineering for most green

alga thanks to its simple genome. In fact, C. reinhardtii was the first engineered

algal species that was studied for commercial purposes, and it was stated that more recombinant proteins have been expressed in C. reinhardtii than the rest of

all examined algal species (Radakovits et al., 2010). Moreover, studies on these

microalgae have also managed to expose the molecular mechanisms behind algal

lipid and hydrogen metabolism. This unicellular alga accumulates large amount

of energy rich molecules such as triacylglycerols (TAGs) under unfavorable

conditions (Nitrogen starvation). These are then converted to biodiesel by trans- esterification reactions (Lenka et al., 2016). However, there are several factors

that make microalgae based biodiesel production economically and

commercially unfeasible. Low light penetrating dense culture, lack of effective extraction methods, and difficulties in harvesting biomass at large scale all lead

to lower oil content (Bellou et al., 2014). Therefore, it is crucial to use metabolic

and genetic engineering to optimize this microalga’s biomass and oil content production to be used for biofuels such as biodiesel (Deng et al., 2012; Deng et

al., 2013). Many believe that ancient algae were responsible for creating crude oil deposits.

Thus, it is clear that using best studied microalgae strains to synthesize biofuels

should be a priority among other alternatives (Pandey et al., 2014). In this bibliographic review, we will be discussing some of metabolic applications to

enhance Chlamydomonas reinhardtii lipid production as well as basic methods

for cultivating and harvesting its biomass. We will also point out the basic principles of extracting oil from cells and trans- esterification reactions, as well

as essential properties that makes microalgae oil appropriate for biofuel

applications.

This bibliographic review gives a concise overview of cultivation of microalgae for the production of biofuel. Chlamydomonas

reinhardtii is one of the best microalgae that can be used for producing biofuel because of its simplicity of genome that allows it to be

easily manipulated by genetic engineering for abundant growth. C. reinhardtii produces lipids under nitrogen deficient condition, which

contains many triacylglycerols (TAGs) that influences the metabolism to a great extent and used for membrane remodeling. Several

genes such as citrate synthase and DGAT2 involved in this mechanism were manipulated by engineering metabolic pathways to

maximize lipid production under nitrogen deprived conditions. Additionally, C. reinhardtii can be efficiently cultivated and grown in

closed photobioreactors, flat plate, tubular or vertical column with protected CO2 rather than open pond systems. In addition, C.

reinhardtii can be harvested by several methods including Tris-Acetate Phosphate Pluronic (TAPP) medium method. Compared to

conventional diesel, microalgae based biofuel, which is produced by trans-esterfication reactions, has a higher pour point temperature

but similar properties. Contrasting Energy-efficiency ratio (EER) values of C. reinhardtii with those of other crops (Jatropha, palm oil

and sunflower) is a crucial process before commercialization. The environmental impacts of microalgae are also beneficial in a way it

can treat wastewater containing large amounts of nutrients such as nitrogen and phosphorus, which are beneficial for algae cultivation

and produce bio-oil. However, this is a risky proposition as it can cause eutrophication and hypoxia in the ponds. Nevertheless, further

research on bio-fuel production from C. reinhardtii must be carried out.

ARTICLE INFO

Received 5. 7. 2018

Revised 24. 4. 2019

Accepted 26. 4. 2019

Published 1. 10. 2019

Regular article

doi: 10.15414/jmbfs.2019.9.2.273-279

J Microbiol Biotech Food Sci / Asmaa Missoum 2019 : 9 (2) 273-279

274

BIOFUELS FROM CHLAMYDOMONAS REINHARDTII

I. Metabolic Engineering and Applications

Recent research in transcriptomics and metabolomics has managed to give better

insights regarding molecular mechanisms involved in TAG synthesis. First, free

fatty acids are produced in the chloroplast after converting pyruvate to Actyl CoA by pyruvate dehydrogenase (PDH). This is further converted to Malonyl

CoA by acetyl-CoA carboxylase (ACCase), which is used by the enzyme

malonyl-CoA:ACP transacylase (MAT) to synthesize Malonyl ACP (Bellou et

al., 2014). This product is consequently involved in the fatty acid synthesis

cycle. Accordingly, after releasing fatty acids into the cytosol, they are converted into acyl CoA by acyl CoA synthetase, which is then involved in series of

reactions by several enzymes on the endoplasmic reticulum (ER). This leads to

assembly of triacylglycerols (TAGs) as shown in figure 1 (Bellou et al., 2014).

Figure 1 Summarizes lipid biosynthesis processes in Chlamydomonas

reinhardtii. GPAT, glycerol-3-phosphate acyltransferase; LPAAT, lyso-

phosphatidic acid acyltransferase; LPAT, lyso-phosphatidylcholine

acyltransferase; DAGAT, diacylglycerol acyltransferase (Bellou et al., 2014).

After selecting strains with maximal TAGs production, out of few options

investigated recently to enhance production of TAGs is to knockdown of the

mRNA expression of the phosphoenolpyruvate carboxylase isoform 1 (CrPEPC1)

gene by RNA interference. In this study, TAGs levels have increased by 20 %

(Deng et al., 2014). During Nitrogen deprivation conditions, genes involved in

triacylglycerol (TAG) biosynthesis are up-regulated whereas those involved in

photosynthesis, protein biosynthesis, and tricarboxylic (TCA) cycle are down-

regulated. Phosphoenolpyruvate carboxylase (PEPC) is an enzyme that catalyzes

the formation of oxaloacetate (OAA) from phosphoenolpyruvate (PEP). This then

enters the TCA cycle to provide the substrate for protein synthesis (Deng et al.,

2014). As mentioned earlier, acetyl-CoA carboxylase (ACCase) catalyzes the

formation of malonyl-CoA from acetyl coenzyme A and consequently enters the

fatty acid synthesis pathway. Therefore, the carbon flux from glycolysis

synthesizes fatty acids or proteins depending on the activities of PEPC and

ACCase as illustrated in figure 2A (Deng et al., 2013).

Figure 2 A Summarizes the distribution of carbon flow between protein and lipid synthesis pathways (Deng et al., 2014). B Summarizes the TCA cycle in which “1”

refers to citrate synthase (Deng et al., 2013).

Given that both lipid and protein biosynthesis pathways share the same

substrate; the deceased consumption of phosphoenolpyruvate of protein pathway helped to increase the final product (TAGs) in the lipid pathway. Thus, the

downregualtion of PEPC using RNAi has lead to a decrease in carbon flow into

the TCA cycle (Deng et al., 2014). Similarly, another way of maximizing TAGs production is by the knockdown of

citrate synthase gene. This gene, also known as ”CrCIS”, codes for the enzyme citrate synthase. Located in the mitochondria, the main function of citrate

synthase in the TCA cycle is to catalyze acetyl CoA reaction to Citroyl CoA

(See figure 2B). In this study, it was reported that TAGs levels were increased by 169.5% after silencing CrCIS using RNA interference. Thus, when the

respective enzyme is not synthesized, acetyl CoA will accumulate as the TCA

cycle is blocked and will be used instead in the lipid biosynthesis pathway. Moreover, silencing of this gene did not slow down cell proliferation (Deng et

al., 2013).

TAGs levels can be also increased by over expressing genes CrDGAT2-1 and

CrDGAT2-5. These are two of the five homologous genes responsible in coding

for DGAT (diacylglycerol acyltransferase) enzymes of different families. These enzymes are involved in assembling TAGs (Final step in the lipid biosynthesis

pathway) by covalently linking diacylglycerols (DAGs) to long-chain fatty acyl-

CoAs. The over expression of CrDGAT2-1 and CrDGAT2-5 genes has lead to 49.75 and 59.01% increase in lipid content respectively (Deng et al., 2012).

Moreover, it important to keep in mind that in previously mentioned studies, Chlamydomonas reinhardtii accumulated more TAGs content under nitrogen

deprivation conditions. These conditions influence the metabolism to a great

extent as acetate is directly funneled into fatty acid biosynthesis and further fatty acids are synthesized by membrane remodeling (Deng et al., 2013). Moreover,

the breakdown of intracellular stores is a useful source of carbon for nitrogen re-

assimilation. Glycolysis genes are also up regulated regardless the decrease in photosynthetic carbon fixation. This indicates that parts of the carbon from

starch are utilized for fatty acid biosynthesis (Deng et al., 2013). However,

nitrogen deprivation also causes thylakoids/chloroplast breakdown, and thus

J Microbiol Biotech Food Sci / Asmaa Missoum 2019 : 9 (2) 273-279

275

almost completely prevents cell growth. Another current study showed that TAGs also accumulated under after phosphorus limitation conditions without

affecting thylakoid membranes (Lenka et al., 2016). Latest research confirms

that supplementing growth culture with 4 g/L sodium acetate counters these effects the result in biomass reduction and increases fatty acids yield by 93.0%

when combined with both nitrogen and phosphorous deficiency (Yang et al.,

2018a). Recently, new strategies were employed to maximise lipid productivity. Using

ptxD gene expression from Pseudomonas stutzeri strain WM88, C.

reinhardtii was capable of using phosphite as its only phosphorus source. This gene encodes for the enzyme phosphite oxidoreductase, which oxidizes

phosphite into phosphate by NAD as a cofactor. This transgenic C. reinhardtii also outcompeted other microalgae and natural contaminants present

in the system, meaning that there is no need to use antibiotics and herbicides for

sterile conditions. This strategy was proven useful for both open pond systems and closed photobioreactors (Loera‐Quezada et al., 2016).

Another way of optimizing lipid biosynthesis in C. reinhardtii is through

induced regulation of two kinases: c-Jun N-terminal kinase (JNK) and extracellular signal regulated kinase (ERK). Under osmotic stress promoted by

1M

NaCl, ERK activation by C6 ceramide and lipid production has augmented by

1.44 and 1.7 fold, respectively. In contrast, inhibition of JNK pathway using

SP600125 has resulted in 40.53% decrease cell growth at the same cultural

conditions (Yang et al., 2018b). The mechanisms of these two kinases under osmotic stress and the effect of biochemical regulations from all experiments in

the study are summerised in figure 3.

Figure 3 Summary of biochemical pathways involved in lipid biosynthesis and

cell growth (yang et al., 2018b).

The microalga C. reinhardtii contains 17 bZIP (basic leucine-region zipper)

transcription factors that are crucial in regulating stress responses which were

identified in a novel study. Also known as CrebZIP, they could play a significant

role in lipid accumulation. This was demonstrated under salt stress conditions

promoted by 150 mM NaCl treatment, which increased lipid content from 0.284

± 0.029 to 0.437 ± 0.012 g/g after 48 hours (Ji et al., 2018). Using quantitative

real time PCR analysis, it was found that only six CrebZIP were expressed such

that the genes CrebZIP4, 5, and 13 were down-regulated, whereas genes

CrebZIP10, 11, and 16 were up-regulated. Therefore, it is possible to exploit the

regulatory mechanisms of these CrebZIP genes in the future studies to engineer

more efficient C. reinhardtii strains, which will eventually enhance biomass and

biofuel production (Ji et al., 2018).

II. Cultivation and Biomass Harvesting

Chlamydomonas reinhardtii is grown in either open pond systems or in large

vessels called photo-bioreactors. These must have CO2 and external light supplies, as well as large surface areas to guarantee enough light diffusion and

CO2 in the media. In open pond systems, ponds are constructed in shallow

dimension to allow the microalgae to use more light as possible (Park et al.,

2011). However, these require large areas of lands and because they are open,

CO2 is lost to the atmosphere. Moreover, uncontrolled growth environmental

Figure 4 A Shows stages of coagulation of microalgal cells (Salim et al., 2010). B Summarizes two mechanisms of flocculation: bridging and patching (Bleeke

et al., 2015). factors such as temperature might lead to contamination, poor light diffusion,

and evaporation (Park et al., 2011).

In closed photo-bioreactors, all conditions are controlled. This gives advantages as undisclosed CO2 is not lost to the atmosphere and the partial pressure is easily

maintained. One commonly used type of these systems is the vertical tubular

photo-bioreactor. These are made of transparent tubes that allow more light penetration. At the bottom of each tube, a sparger is attached to convert sparged

gas into tiny bubbles and removed produced O2 during photosynthesis. Mixing in these systems is achieved mechanically by a paddlewheel and using gas flows

respectively. This helps to distribute metabolite, cells, and gases homogenously

in the media. It is also important to consider that the optimum conditions for cultivation varies for one strain to another and depends on which system is used.

Generally, keeping pH at 7.5 and using moderate CO2 levels will enhance

cultivation (Pandey et al., 2014). The first step of the microalgae harvesting is screening. Here, the biomass is

introduced into a screen in order to filter anything else and capture only

microalgae cells. This can be accomplished by various methods including the

use of micro-strainers or vibrating screens. The following step requires the use

of chemicals that induce coagulation and flocculation (Salim et al., 2010). This

causes microalgae cells to aggregate into clamps that can be filtered easily from the medium (Figure 4A). In order to achieve coagulation, two types of chemicals

are used: inorganic coagulants such as metal ions Fe3+ and Al3+ from Aluminum

and Ferric sulphates, and long chain organic coagulants like poly-electrolytes and natural polymeric substances. The former type are cations that bind to

negatively charged microalgae particles and thus neutralize the environment

such that they do not repel each other anymore and therefore clump together. Similarly, flocculation using polymers causes cells to bind to positively charged

polymers. If they bind partly, the unoccupied spaces of polymers can bind other

cells and consequently bridge them. However, if they bind completely, they patch to the surface creating local positive charges that can attract other

microalgae cells resulting in flocculation (Bleeke et al., 2015). This is illustrated

in figure 4B. In order to harvest the small sized microalgae such as C. reinhardtii, a method

called floatation is used. Its main principle is based on that fact that when solid

particles attach to air bubbles, they result in flocs floating the broth or liquid

surface because of buoyancy (Chen et al., 2011). This method costs a lot as it

requires bubbling air through a column, using maximum amount of coagulants,

and adjusting other factors such as pH. Bulk harvesting methods (flocculation) uses low energy that can be cost effective to harvest C. reinhardtii. Iron chloride

(FeCl3), aluminium sulphate (Al2(SO4)3), and iron sulphate (Fe2(SO4)3) are

conventional flocculants (Brennan and Owende, 2010) that transfer microalgal cells to slurry to stay out of the cultivation medium. Filtration can then release

water from the slurry to concentrate the amount of microalgal slurry.

Development of biodegradable and harmless organic flocculants can be used for harvesting algae (Suali and Sarbatly, 2012).

Gravity sedimentation is another method to separate microalgal cells such that

this time they settle to the bottom out of the fluid responding to forces of gravity. However, because the microalgae have small size, the settling rate is too low and

slow (Bux, 2013). For this reason, this rate is increased by centrifugational

force. Centrifugation is very effective as it was reported that it results in 90-100% harvesting efficiency but it is also cost and energy intensive (Pandey et

al., 2014).

J Microbiol Biotech Food Sci / Asmaa Missoum 2019 : 9 (2) 273-279

276

Although research on improving previously mentioned harvesting methods continues, other new strategies and devices are invented. Recently, researchers

cultured Chlamydomonas reinhardtii in a Tris-Acetate Phosphate Pluronic

medium, also known as TAPP successfully without affecting the productivity (Estimeet et al., 2017). This medium undergoes a thermoreversible sol-gel

transition, meaning that after culturing microalgae in the TAPP medium in a

solution phase at 15 °C, the temperature is increased at 22 °C to cause medium gelation and trap microalgal cells as illustrated in figure 5 .

Figure 5 Summary of cultivation and harvesting Chlamydomonas reinhardtii

cells stages using thermo-reversible gel and Tris-Acetate Phosphate Pluronic

(TAPP) medium (Estimeet et al., 2017).

After the cultivation period, the temperature is lowered to 15 °C allowing

microalgal clusters to gravimetrically settle at the bottom. Finally, the

temperature was increased to jellify the supernatant and harvesting of the

microalgae simply involved scraping microalgal flocs off the TAPP surface. The

whole process is repeatable and takes only 2 hours without further requirement of

additional materials. Another advantage of the TAPP medium is that it can be re-

used and recycled medium for re-cultivation of microalgae after the harvesting

process (Estime et al., 2017).

In order to improve biomass production, old concepts of crops enhancement can

be used instead of DNA genetic techniques that are used to boost lipid

production. In a novel study, diploidy was induced in C. reinhardtii by treating

the haploid cells with colcemid, a microtubule inhibitor. Then, two diploid

strains, CMD ex1 and CMD ex4, were selected. These coped well when exposed

to cold, nutritional, and oxidative stresses, as well as accumulated biomass and

Fatty acids yield by two fold when compared to the control (Kwak et al., 2017).

Another strategy involves the use of microalgae growth promoting bacteria

(MGPB) as ‘probiotics’. These were obtained from wastewater effluent samples

in Yamanashi, Japan and were tested on three different microalgae including C.

reinhardtii. The bacteria enhanced their host’s biomass production by nearly 1.5

fold after 7 days. Using quantitative PCR and 16S rRNA gene analysis, the

classes of bacteria were identified as Alphaproteobacteria, Betaproteobacteria,

Sphingobacteriia and Saprospirae (Toyama et al., 2018).

III. Preparation of Chlamydomonas reinhardtii Oil as Biofuel

This is an important multi step process that depends heavily on how biomass

was previously harvested. For this reason, selecting best method is very crucial as it consequently affects biofuel production. First, the biomass is dried to avoid

moisture interference with solvents. Cells are then disrupted by various methods

such as squeezing cells under high pressure (Expeller pressing) or using beads (Bead beating) in order to get intracellular contents to the solvent medium.

Afterwards, algal oil is extracted by many methods (Bellou et al., 2014). One of

these methods is solvent extraction as lipids are soluble in organic solvents. Lipids’ solubility plays a major role as each value depends on the type of solvent

that is more suitable to it. For example, polar lipids such as Phospholipids

dissolve in polar solvents and non-polar lipids such as triglycerides dissolve non-polar solvents (Lam and Lee, 2012). Enzymatic treatment could be also used to

break down cell wall with water acting as solvent and thus oil content separates

during the process. However, this is not feasible for large scale application. Other methods including osmotic shock, pulse electric field technology, and

hydrothermal liquefaction are highly expensive and necessitate further optimization efforts (Suali and Sarbatly, 2012).

Transesterification is a reversible process where one mole of triacylglycerides is

reacted with three moles of alcohol, usually methanol or ethanol, as well as adding a catalyst (See figure 6) to synthesize glycerol as byproduct and fatty

acid methyl esters (FAME). The latter are then used as biodiesel. In an acid

catalyzed transesterification, the reaction is carried at 80°C at a slow rate requiring high amounts of alcohol. One example of the catalyst used is

H2SO4/HCl. However, in an alkali catalyzed one, it is completed at 60°C in 90

minutes requiring the use of potassium and sodium hydroxides as catalysts (Pandey et al., 2014). This process is 4000 times faster but water removal is a

crucial step before beginning as it causes base hydrolysis. Another type of transesterification reaction is one that is catalyzed by lipases enzymes as they

convert all triglycerides into biodiesel. This process is extremely unfeasible as it

requires complex processing (Brennan and Owende, 2010).

Figure 6 Summary of acid or base catalyzed trans-esterfication process of

triacylglycerides (TAGs) with methanol giving glycerol and fatty acid methyl esters

(FAME) (Pandey et al., 2014). In order for fatty acid methyl esters to be used as biodiesel, certain

characteristics such as iodine and acid number, flash and pour points, and

kinematic viscosity must be examined. The iodine number refers to the unsaturation degree and acid number indicates the corrosives of oil. The flash

point is the lowest temperature at which oil vaporizes to potentially form

ignitable mixtures and that of biodiesel is much higher than conventional diesel (Pandey et al., 2014). Additionally, pour point is the lowest temperature at

which oil loses its flow properties and becomes semi-solid. This is a vital

property to qualify microalgae oil as a diesel because its pour point is slightly higher than that of conventional diesel. This is useful in countries with tropical

hot weather such as India. Other important characteristics include heating value

and cetane number. The former is defined as the energy released as heat when the compound undergoes combustion and the latter refers to ignition quality

(Suali and Sarbatly, 2012). All properties of both microalgae biodiesel and

conventional diesel are compared in summarized in table 1 below, suggesting that it is a good candidate for biofuel applications (Brennan and Owende,

2010).

Table 1 Comparison between microalgae biodiesel and conventional diesel fuel

properties (Pandey, 2014).

S. No. Fuel Property Biodiesel Standards

(ASTM)

Microalgae

Biodiesel

Diesel Fuel

(ASTM)

1 Acid number

(mg KOH/g of oil)

˂0.5 0.42 0.7-1.0

2 Iodine value

(g I/100 g of oil)

˂25 (efficient fuel) 19.0 120

3 Specific gravity (g/cm3) 0.85-0.90 0.85 0.82-0.90

4 Density (g/cm3) 0.88 0.85 0.86-0.90

5 Kinematic viscosity

(mm2/s)

1.9-6.0 2.0-4.5 3.5

6 Heating value (MJ/kg) 44 37 42.2

7 Flash point (◦C) 130 ˃130 ˃62

8 Pour point (◦C) -11.6 -6 -16

9 Cetane number 47 46 60

IV. Scale up and Commercialization

As mentioned earlier, cultivating C. reinhardtii for biodiesel production has

many advantages. These include the fact that it is fast growing and have 100

times higher reproducibility than land plants. In addition, it accumulates lipids as 75% of its total weight, and unlike plants, agricultural lands are not needed to

cultivate it. Large-scale cultivation of C. reinhardtii faces numerous technical challenges that slow down the commercialization of renewable microalgae

feedstock for producing biodiesel (Lam and Lee, 2014). These consist of

upstream processes such as selecting C. reinhardtii strains for cultivation,

nutrient sources, minimum energy input for closed photo-bioreactors, reusing

water and sensitivity of microalgae for surrounding the environment. Other

challenges include downstream processes such as harvesting and drying C. reinhardtii cells, conversion of microalgal biodiesel with good quality, and

effective techniques for lipid extraction. Finding solutions for these challenges

J Microbiol Biotech Food Sci / Asmaa Missoum 2019 : 9 (2) 273-279

277

could enhance the economic feasibility of microalgae-based biodiesel production (Lam and Lee, 2014).

A minimum input of energy and cost-effective protocols are the first challenges

to consider when commercializing algal biofuels. Cultivation systems need to have large areas of light, utilize optimal gas-liquid transfer, low or no

contamination and must be easy to operate (Xu et al., 2009). The preferred

technique of cultivating C. reinhardtii is phototropic cultivation, and it requires a high intensity of sunlight and it recaptures CO2 from flue gases. This method has

limitations if sunlight is not very radiant in case of temperate countries (Lam

and Lee, 2012). Moreover, phototropic C. reinhardtii can grow in open pond system and closed photo-bioreactor. Considering the issue of energy balance for

producing microalgal biofuels, when it is cultivated in closed photo-bioreactors, studies by life-cycle assessments (LCAs) of C. reinhardtii biodiesel production

showed that there was a negative energy balance in cultivation (Jorquera et al.,

2010; Razon and Tan, 2011; Stephenson et al., 2010). Energy-efficiency ratio (EER) can improve the energy conversion efficiency of

algal biofuel production before commercialization. The EER is the ratio that

represents total output of energy to total input of energy. If the ratio is higher than 1, then net positive energy is produced. Table 2 shows a study on EER for

biodiesel originated from Jatropha, palm oil, sunflower, and Chlamydomonas

reinhardtii. The values are an estimated indication based on different

assumptions from studies of the LCA. In Table 2, microalgae (C. reinhardtii)

contains the highest EER out of the feedstock crops used, which means the

energy required for microalgae cultivation requires high energy input to produce biofuel. Moreover, the cultivation of C. reinhardtii for producing biodiesel may

create a progressive energy output (Lam and Lee, 2012).

Table 2 Energy-efficiency Ratio (EER) for Algae and crops (Lam and Lee,

2012).

Feedstock EER Comment

Jatropha 3.40 Included biogas production

Palm Oil 3.53 Included co-product production

Sunflower 3.50 Based on Chilean conditions

Chlamydomonas reinhardtii

4.34 Low nitrogen culture and biomass are not droed for

extraction

Closed photo-bioreactors are widely used for C. reinhardtii cultivation. They are

designed to ensure that the microalgae will be produced at optimal conditions

with high stability with a minimum level of contamination. Water sources are re-

utilized for cultivation cycles. Physiological and biological factors such as pH, concentration of CO2, and nutrient level can optimize cultivation of C.

reinhardtii strains in closed photo-bioreactors. A closed photo-bioreactor always

produces high biomass productivity of 0.05–3.8 g/L/day with the mass ratio of 0.00035–0.027 of C. reinhardtii to water in 7 days when conditions of the

cultivation and algal strains are controlled and optimized (Brennan and

Owende, 2010; Lam and Lee, 2012). Examples of closed photo-bioreactors are flat plate, tubular and vertical column.

Figure 6 outlines the structures of the three examples. Flat plate is a rectangular

structure that has a large area of radiating sunlight and utilizing solar energy and uses a gas pipe that contains low dissolved oxygen, although it is difficult in

controlling temperature of culture (Lam and Lee, 2012). Tubular method

consists of looped transparent tubes that has a large area same as flat plates that uses sunlight for energy and processing, they produce a high biomass of the

microalgae and reduce cell damage, although mixing can be challenging when

products appear in whole tube, also long tubes are used so cultivation requires a

vast area. The vertical column is a vertically rearranged cylinder column that

have a high mass area that mixes C. reinhardtii very well for cultivation, its

columns are made of transparent glass and the method is compact and easy to operate, also it is inexpensive (Lam and Lee, 2012).

Table 3 Examples of closed photobioreactors and their structures (Chen et al., 2011).

Closed Photo-Bioreactor Structure

Flat plate

Tubular

Vertical column

There are upstream and downstream processes of C. reinhardtii cultivation. One of the upstream processes of algal cultivation includes water reusability and

footprint (WF). Water footprint is when an area contains the total amount of

fresh water to form a product for consumers (Mata et al., 2010). Production of biofuel requires 1 L of microalgal biodiesel and 3000 L of water, and water is

recycled for cultivation of microalgae to lessen the water demand. Water

demand is the difference between the required water for cultivation and the annual precipitation (Xu et al., 2009). If lipid productivity is low in a C.

reinhardtii strain for biofuel production then area of cultivation is highly

required. Lipid productivity in microalgae to produce biofuel was discussed earlier. C. reinhardtii are reproduced in water when in culture medium; non-

cellular water of 200kg is needed for growing microalgae for cultivation (Xu et

al., 2009).

One of the downstream processes of C. reinhardtii cultivation is harvesting the

microalgal biomass, which is separating algae from water to produce biofuel. The first step is bulk harvesting, to separate the microalgae from suspended bulk in

gravity sedimentation, flocculation, and flotation and the second step is to thicken

and concentrate the microalgae after harvesting bulk by filtration and centrifugation as discussed earlier (Mata et al., 2010). It is difficult to harvest the

microalgal cells and suspend them in water, because they have a small size of 1–

20 mm. Water of 73 tonnes need to be treated when harvesting the microalgal biomass of 1 tonne for cultivation. This develops effective C. reinhardtii

harvesting for strengthening the commercialization of microalgal biofuel

production (Mata et al., 2010).

V. Economics

There are three major contributions to production cost: depreciation, raw

materials and utilities, and labor and supervision. Figure 7 shows a flowchart of the capacity of the production and parameters of the distinctive developed

operations. It provides the available type and size as well as energy and mass of

equipment (Acien et al., 2014).

Figure 7 Flowchart of the cost analysis for C. reinhardtii production (Acien et

al., 2014).

According to the flow chart, the required raw materials are calculated from mass balances. The utilities and consumption is calculated from use of power and

water. The cost of raw materials includes the suppliers’ market values and facility

transport. With regard to power, the cost of electricity can vary according to

consumption and energy required; therefore, a detailed analysis of different

suppliers is recommended (Acien et al., 2014).

Cities with high pollution can generate high amounts of wastewater and it provides as a fertilizer. Untreated or treated wastewater can replace fertilizers and

are cost effective; they also form a revenue stream to compensate the amount of

bio-crude. Reducing the nutrient level satisfactorily minimized pollution of wastewater in China lakes and rivers, and for a while the lake will purify itself.

Consequently, treating wastewater for treatment plant can produce growing diatoms. Treatment plant converts organic nutrients to inorganic ones.

Economically viable products can be made from integrated system of growing

diatoms in producing fuel. Fertilizers are the most expensive cost of C. reinhardtii based biofuels (Acien et al., 2014; Mata et al., 2010].

Moreover, 5kt per year of C. reinhardtii is produced internationally in markets.

The price ranges from €10–300/kg and the market size is 10–50 kt/year. Moreover, with high salary levels cost of labor of workers that are managing and

supervising the process increases. For establishing cost production of biomass of

C. reinhardtii used for biofuel, modifying the cost of the scaled equipment is used and multiplying the direct cost by the suitable factor of 10 raises the

capacity, in which the formula can be simplified as CostB=CostA (SizeB /

SizeA) scale-up factor (Acien et al., 2014).

VI. Environmental Impacts

J Microbiol Biotech Food Sci / Asmaa Missoum 2019 : 9 (2) 273-279

278

Environmental impacts of C. reinhardtii biofuels have its benefits and drawbacks. Depending on the input resources and the cultivation, C. reinhardtii

biofuels could either damage quality of water or enhance it. The microalgae

remove nutrients from waste by their potential water-quality that reduces eutrophication and runoff of herbicides and insecticides. Therfore, it is more

beneficial in their water quality than corn-based ethanol and soybean-based

biodiesel. The effects of water quality depend on the amount of nutrient of the microalgal culture medium, whether systems of feedstock are covered from

heavy rain. Floods or culture fluid leakage to groundwater can happen if the

reliability of the pond is compromised although these downfalls can be prevented with good managing of the culture processes (Bellou et al., 2014).

In C. reinhardtii cultivation, water can be re-used for easier cultivation. The liquid waste can be re-used to form biogas from the anaerobic respiration (Davis

et al., 2011). In addition, released waters from C. reinhardtii cultivation aquifers

are saline. The Clean Water Act and National Pollutant Discharge Elimination System can operate facilities of the microalgae cultivation. Releasing harvest

water for terrestrial crops can be against the law for using it for biofuel factories.

Moreover, leaching to groundwater could cause matters of C. reinhardtii cultures to be lost this can be caused by wind and high precipitation. Unintentional spills

of cultivation water can be caused by environmental hazards such as hurricane,

tornadoes or even earthquakes. Producers can deal with unintentional spills of

cultivation water by defensive measures for predicting when hazardous weather

events would take place, so that it does not affect their income (Bellou et al.,

2014). Large amounts of nitrogen and phosphorus are needed for a high yield of large-

scale cultivation of C. reinhardtii. Accidental spills of cultivation water causes

eutrophication of freshwaters and marine waters. Eutrophication signifies microalgal growth caused by presence of high concentrations of nitrogen and

phosphorus in ponds. When C. reinhardtii decompose in the ponds generating

high organic matter will lower the oxygen level in the water, which can lead to hypoxia in the ponds (Rabalais et al., 2009; Smith and Schindler, 2009). In

some cases, eutrophication-induced changes could be difficult or impossible to

reverse if alternative stable states can occur in the affected ecosystem (Carpenter, 2005).

C. reinhardtii have been also involved in treating wastewater, where they provide

photosynthetically produced oxygen for the bacteria, which breakdown organic compounds present in the waste. High rate algal ponds (HRAPs) treat agricultural

wastewater and growth of photosynthetic microalgae (Park et al., 2011). HRAPs

remove nitrogen and phosphorus from the wastewater by algae. Additionally, in

wastewater treatment, phosphorus is precipitated by insoluble solid (aluminum or

iron-based coagulants) or it is unprotected from the water by microbial activity.

The phosphorus when developed forms a sludge fertilizer. It is a beneficial process in lab experiments to recycle nutrients from wastewater and use them for

further C. reinhardtii production (Sturm and Lamer, 2011).

CONCLUSION

Chlamydomonas reinhardtii is essential by many aspects for producing biofuels. C. reinhardtii is easily used for genetic engineering from its simple

genome and produces lipids for biofuel. The partial pressures are constant for

sustainability. Also C. reinhardtii compared to other conventional diesel have better properties such as a high point temperature for greater biofuel

production. Additionally, the microalga has a higher energy-efficiency ratio

(EER) than other crops used for biofuels for better cultivation and efficiently producing biofuel. Recommendations of producing biofuels from microalgae

are using biodiesel to exchange fossil-based petroleum, because it is easier for

lipid extraction to produce it, using low amount of oil for scale

commercialization of products of biofuels and having efficient technologies for

harvesting microalgae is also recommended. Finally, sustaining algal biofuels

is very beneficial for production of biofuels from these factors: land use, balance of carbon, nutrient sources, balance of energy and water.

REFERENCES

Acien F.G, Fernandez J.M, Molina-Grima E. (2014). Economics of Microalgae Biomass Production. Elsevier B.V. Chapter 14. 314-316.

https://doi.org/10.1016/B978-0-444-59558-4.00014-0

Bellou S, Baeshen M, Elazzazy A, Aggeli D, Sayegh F, Aggelis G. (2014). Microalgal lipids biochemistry and biotechnological perspectives. Biotechnol

Adv. 32: 1476–93. https://doi.org/10.1016/j.biotechadv.2014.10.003

Bleeke, F., Milas, M., Winckelmann, D., & Klöck, G. (2015). Optimization of freshwater microalgal biomass harvest using polymeric

flocculants. International Aquatic Research, 7(3), 235-244.

https://doi.org/10.1007/s40071-015-0108-8 Brennan, L., Owende, P. (2010). Biofuels from microalgae - A review of

technologies for production, processing, and extractions of biofuels and co-

products. Renew. Sustain. Energy Rev. 14, 557–577. https://doi.org/10.1016/j.rser.2009.10.009

Bux, F. (2013). Biotechnological applications of microalgae: biodiesel and value-

added products. Boca Raton, FL: CRC Press, Taylor & Francis Group.

https://doi.org/10.1111/jpy.12233 Carpenter, S.R. (2005). Eutrophication of aquatic ecosystems: Bistability and soil

phosphorus. Proceedings of the National Academy of Sciences of the United

States of America 102(29): 10002-10005. https://doi.org/10.1073/pnas.0503959102

Chen, C.Y., Yeh, K.L., Aisyah, R., Lee, D.J., Chang, J.S., (2011). Cultivation,

photobioreactor design and harvesting of microalgae for biodiesel production: A critical review. Bioresour. Technol. 102, 71–81.

https://doi.org/10.1016/j.biortech.2010.06.159

Estime, B., Ren, D., & Sureshkumar, R. (2017). Cultivation and energy efficient harvesting of microalgae using thermoreversible sol-gel transition. Scientific

Reports, 7, 40725. http://doi.org/10.1038/srep40725 Davis, R., A. Aden, & P.T. Pienkos. (2011). Techno-economic analysis of

autotrophic microalgae for fuel production. Applied Energy 88(10): 3524-3531.

https://doi.org/10.1016/j.apenergy.2011.04.018 Deng, X., Gu, B., Li, Y., Hu, X., Guo, J., & Fei, X. (2012). The Roles of acyl-

CoA: Diacylglycerol Acyltransferase 2 Genes in the Biosynthesis of

Triacylglycerols by the Green Algae Chlamydomonas reinhardtii. Molecular Plant, 5(4), 945-947. https://doi.org/10.1093/mp/sss040

Deng, X., Cai, J., & Fei, X. (2013). Effect of the expression and knockdown of

citrate synthase gene on carbon flux during triacylglycerol biosynthesis by green

algae Chlamydomonas reinhardtii. BMC Biochemistry, 14, 38.

http://doi.org/10.1186/1471-2091-14-38

Deng, X., Cai, J., Li, Y., & Fei, X. (2014). Expression and knockdown of the PEPC1 gene affect carbon flux in the biosynthesis of triacylglycerols by the

green alga Chlamydomonas reinhardtii. Biotechnology Letters, 36(11), 2199-

2208. https://doi.org/10.1007/s10529-014-1593-3 Ji, C., Mao, X., Hao, J., Wang, X., Xue, J., Cui, H., & Li, R. (2018). Analysis of

bZIP Transcription Factor Family and Their Expressions under Salt Stress

in Chlamydomonas reinhardtii. International Journal of Molecular Sciences, 19(9), 2800. http://doi.org/10.3390/ijms19092800

Jorquera, O., Kiperstok, A., Sales, E.A., Embiruc ̧u, M., Ghirardi, M.L., (2010).

Comparative energy life-cycle analyses of microalgal biomass production in open ponds and photobioreactors. Bioresour. Technol. 101, 1406–1413.

https://doi.org/10.1016/j.biortech.2009.09.038

Kwak, M., Park, W., Shin, S., Koh, H., Lee, B., Jeong, B., & Chang, Y. K. (2017). Improvement of biomass and lipid yield under stress conditions by using

diploid strains of Chlamydomonas reinhardtii. Algal Research, 26, 180-189.

https://doi.org/10.1016/j.algal.2017.07.027

Lam, M. K., Lee, K.T. (2012). Microalgae biofuels: A critical review of issues,

problems and the way forward. Biotechnol. Adv. 30, 673–690.

https://doi.org/10.1016/j.biotechadv.2011.11.008 Lam M. K, Lee K. T. (2014). Scale-Up and Commercialization of Algal

Cultivation and Biofuel Production. Chap 12. Elsevier B.V. 256-266.

https://doi.org/10.1016/B978-0-444-64192-2.00019-6 Lenka, S. K., Carbonaro, N., Park, R., Miller, S. M., Thorpe, I., & Li, Y. (2016).

Current advances in molecular, biochemical, and computational modeling

analysis of microalgal triacylglycerol biosynthesis. Biotechnology Advances, 34(5), 1046-1063. https://doi.org/10.1016/j.biotechadv.2016.06.004

Loera‐Quezada, M. M., Leyva‐González, M. A., Velázquez‐Juárez, G., Sanchez‐

Calderón, L., Do Nascimento, M., López‐Arredondo, D., & Herrera‐Estrella, L. (2016). A novel genetic engineering platform for the effective management of

biological contaminants for the production of microalgae. Plant Biotechnology

Journal, 14(10), 2066–2076. http://doi.org/10.1111/pbi.12564 Mata, T.M., Martins, A.A., Caetano, N.S. (2010). Microalgae for biodiesel

production and other applications: A review. Renew. Sustain. Energy Rev. 14,

217–232. https://doi.org/10.1016/j.rser.2009.07.020

Pandey, A., Lee, D.J., Chisti, Y., Soccol, C.R. (2014). Biofuels from algae (1st

ed.). Amsterdam: Elsevier. https://doi.org/10.1016/C2012-0-00170-6

Park, J.B.K., R.J. Craggs, and A.N. Shilton. (2011). Wastewater treatment high rate algal ponds for biofuel production. Bioresource Technology 102(1): 35-42.

https://doi.org/10.1016/j.biortech.2010.06.158

Rabalais, N.N., Turner R.E., Díaz R.J., and Justi D. (2009). Global change and eutrophication of coastal waters. ICES Journal of Marine Science 66(7):1528-

1537. https://doi.org/10.1093/icesjms/fsp047 Radakovits, R., Jinkerson, R. E., Darzins, A., & Posewitz, M. C. (2010). Genetic

Engineering of Algae for Enhanced Biofuel Production. Eukaryotic Cell, 9(4),

486-501. https://doi.org/10.1128/ec.00364-09 Razon, L.F., Tan, R.R., (2011). Net energy analysis of the production of biodiesel

and biogas from the microalgae: Haematococcus pluvialis and Nannochloropsis.

Appl. Energy 88, 3507–3514. https://doi.org/10.1016/j.apenergy.2010.12.052 Salim, S., Bosma, R., Vermuë, M. H., & Wijffels, R. H. (2010). Harvesting of

microalgae by bio-flocculation. Journal of Applied Phycology, 23(5), 849-855.

https://doi.org/10.1007/s10811-010-9591-x

Smith, V.H., and D.W. Schindler. (2009). Eutrophication science: Where do we

go from here? Trends in Ecology and Evolution 24(4): 201-207. https://doi.org/10.1016/j.tree.2008.11.009

Stephenson, A.L., Kazamia, E., Dennis, J.S., Howe, C.J., Scott, S.A., Smith,

A.G., (2010). Life-cycle assessment of potential algal biodiesel production in the

J Microbiol Biotech Food Sci / Asmaa Missoum 2019 : 9 (2) 273-279

279

united kingdom: A comparison of raceways and air-lift tubular bioreactors. Energy Fuels 24, 4062–4077. https://doi.org/10.1021/ef1003123

Sturm, B.S.M., and S.L. Lamer. (2011). An energy evaluation of coupling

nutrient removal from wastewater with algal biomass production. Applied Energy 88(10): 3499-3506. https://doi.org/10.1016/j.apenergy.2010.12.056

Suali, E., Sarbatly, R., (2012). Conversion of microalgae to biofuel. Renew.

Sustain. Energy Rev. 16, 4316–4342. https://doi.org/10.1016/j.rser.2012.03.047 Xu, L., Weathers, P.J., Xiong, X.R., Liu, C.Z., (2009). Microalgal bioreactors:

Challenges and opportunities. Eng. Life Sci. 9, 178–189.

https://doi.org/10.1002/elsc.200800111 Yang, L., Chen, J., Qin, S., Zeng, M., Jiang, Y., Hu, L., Wang, J. (2018a).

Growth and lipid accumulation by different nutrients in the microalga Chlamydomonas reinhardtii. Biotechnology for Biofuels, 11, 40.

http://doi.org/10.1186/s13068-018-1041-z

Yang, A., Suh, W. I., Kang, N. K., Lee, B., & Chang, Y. K. (2018b). MAPK/ERK and JNK pathways regulate lipid synthesis and cell growth

of Chlamydomonas reinhardtii under osmotic stress, respectively. Scientific

Reports, 8, 13857. http://doi.org/10.1038/s41598-018-32216-5